leky-latky/cytokiny/40-jednotlive-cytokininy-a-pusobeni

Cytokinin ribosides

• Evaluated in patients with diverse malignancies in 1970s (Mittelman et al. 1975)
• Inspired development of inhibitors of cyclin-dependent kinases
• Olomoucine
• Bohemine
• Roscovitine
• Their analogues (Veselý et al. 1994; Havlíček et al. 1997; Vermeulen et al. 2002; Kryštof et al. 2002) [1]

iPR and N6-benzyladenosine (BAR)

• Studied traditionally as anti-cancer agents
• Possess cytoprotective activities as well
• Dassano et al. (2014) treatment of several cancer cell lines with iPR or BAR
• Induced robust expression of genes regulated by transcription factor Nrf2
• Transcription of Nrf2 itself was upregulated
• Treatment with cytokinin ribosides
• Decreased the basal levels of ROS
• Increased the resistance of the cells to pro-oxidative insults [1]

• Not only iPR but also other cytotoxic ribosides
• Kinetin riboside (KR)
• In low micromolar concentrations induce Nrf2-dependent gene expression in multiple cell lines [1]

Antiproliferative effects of cytokinins and cytokinin analogues

• Natural cytokinin ribosides iPR, KR, BAR, ortho-topolin riboside (oTR) and N6-(2-hydroxy-3-methoxybenzyl)adenosine (but not their respective bases)
• Reported to exhibit strong cytotoxic effects against a range of human cell lines derived from
• Hematological malignancies
• Solid tumors (Doležal et al. 2007; Voller et al. 2010; Voller et al. 2017 – in press) [1]

• Some cytokinin ribosides are active at
• Submicromolar (against some leukemia)
• Micromolar concentrations (against other leukemia, adherent cells) [1]

• Toxicity of tZR and cis-zeatin riboside
• Differ from iPR by hydroxylation of the isoprenoid side-chain
• Is very limited (Ishii et al. 2002; Rattan and Sodagam 2005; Voller et al. 2010) [1]

• Isomers of oTR with a hydroxy group in either the meta- or para-position of the phenyl ring
• Do not show significant toxicity (Voller et al. 2010) [1]

• In leukemia cell lines
• Cytokinin ribosides induce rapid apoptosis (Mlejnek and Kuglík 2000; Ishii et al. 2002; Voller et al. 2017 – in press)
• Cell death is preceded by ATP depletion
• Exception is N6-(2-hydroxy-3-methoxybenzyl)adenosine
• Apoptosis ensues without marked effects on cell energy levels (Voller et al. 2017 – in press)

• Cytotoxicity of iPR and KR
• Against mammalian cell lines derived from solid tumors
• Reported many times
• Meisel et al. 1998; Griffaut et al. 2004; Laezza et al. 2006; Spinola et al. 2007; Cheong et al. 2009; Laezza et al. 2009; Cabello et al. 2009; Colombo et al. 2009; Laezza et al. 2010; Rajabi et al. 2012; Wang et al. 2012; Ciaglia et al. 2014; Ciaglia et al. 2016 [1]

• Depending on the cell line and cytokinin used, the treatment resulted in
• Apoptosis,
• G1
• G2/M block [1]

• Spectrum of the effects induced by cytokinin ribosides in the tested cell lines included
• ATP depletion
• Genotoxic stress (Cabello et al. 2009)
• JNK activation (Laezza et al. 2009)
• Inhibition of farnesyl-protein transferase activity (Laezza et al. 2006)
• Inhibition of EGFR signaling (Ciaglia et al. 2016)
• Changes in levels of mitochondrial proteins (Cheong et al. 2009) [1]

• Microarray analysis of the effects of iPR (100 µM) on MCF7 and A549 cell lines was published
• IPR induced a set of genes involved in stress induced cell cycle arrest
• PPP1R15A, DNAJB9, DDIT3, and HBP1 (Colombo et al. 2009) [1]

• Cytokinin ribosides
• May also interfere with neoangiogenesis (Pisanti et al. 2014) [1]

• Cytokinin riboside-5’-monophosphates
• Identified as inhibitors of putative oncogene RCL1 (Amiable et al. 2013, Voller et al. 2017 - in press) [1]

• Anticancer activity of iPR, KR, oTR and BAR
• Demonstrated using several animal and xenograft models of cancer (Griffaut et al. 2004; Laezza et al. 2006; Tiedemann et al. 2008; Voller et al. 2010)

• IPR and BAR
• Limited activity against a diverse range of cancers in a small clinical trial (Mittelman et al. 1975)

• Micromolar concentrations of both cytokinin ribosides and cytokinin bases can also induce cell death in plant cell cultures
• Activation of caspase-like proteases
• Fragmentation of DNA (Mlejnek and Procházka 2002; Mlejnek and Doležel 2005)
• Cell death is preceded by depletion of ATP and the production of ROS [1]

• Intracellular conversion of cytokinins to 5’-monophosphates
• Is necessary for their cytotoxic effect
• Concentrations of cytokinins required to produce cytotoxic effects are higher than those found endogenously in plant tissues [1]

• Phosphorylation of cytokinin ribosides
• By adenosine kinase (ADK)
• Requirement for the cytotoxic effect of cytokinin ribosides in both animal (Mlejnek and Doležel 2005; Voller et al. 2010) and plant cells (Mlejnek and Procházka 2002)
• Low affinity to ADK (and possibly other nucleoside kinases)
• May explain the lack of activity of other cytokinin ribosides (Mlejnek and Doležel 2005) and possibly also their analogues with ribose replaced by acyclic polyols (Colombo et al. 2009; Ottria et al. 2009)

• Contrary to other nucleoside analogues that are converted to nucleoside triphosphates
• Dominant metabolites of cytokinin ribosides are their respective riboside monophosphates (Mlejnek and Doležel 2005)
• Cytokinin ribosides have a different mechanism of action than classical antimetabolites
• Therse after phosphorylation, directly interfere with the synthesis of nucleic acids

• Cytoxicity of cytokinin bases and their corresponding ribosides
• Are comparable
• In contrast to human cell lines, plant cells are able to convert both metabolic forms efficiently into riboside-5 ´-monophosphates (Mlejnek and Procházka 2002; Mlejnek and Doležel. 2005) [1]

• Characteristic trait of leukemia cells
• Is blockade of their differentiation into functional mature cells
• Various chemicals acting by diverse mechanisms are able to force malignant cells to undergo terminal differentiation
• Such differentiation therapy could be much safer than regimens based on cytotoxic effects

• Cytokinin bases K, iP, BA and, to lesser degree, also tZ
• Shown to induce granulocytic differentiation of human myeloid leukemia cell line HL-60 (Ishii et al. 2002) derived from the peripheral blood leukocytes of a patient with acute myeloid leukemia
• Activity was mediated by
• Phosphorylation of ERK1/2
• Expression of CEBPD
• Expression of S100P (Ishii et al. 2005a; Ishii et al. 2005b)

• Cytokinin bases
• Induced differentiation at rather high concentrations (25-100 µM)
• Their ribosides caused rapid apoptosis at low micromolar levels

• Treatment with caspase inhibitors
• Shifted the activity of iPR in HL-60 from pro-apoptotic to growth inhibitory and differentiating activity (Ishii et al. 2002)

• Cytokinin ribosides
• Induce Nrf2-dependent transcription (Dassano et al. 2014, our unpublished data)
• Inhibit proteosynthesis
• Decrease of activating phosphorylation of p70 S6 kinase and S6 protein
• Activate autophagy
• Increase in levels of LC3-II (unpublished data) [1]
• Implicated in cytoprotection and aging prevention (Bruns et al. 2015; Madeo et al. 2015)

• If cytokinin bases are metabolized into respective ribosides/ribotides
• In quantities sufficient to activate those stress response pathways
• Could act as hormetin precursors - prohormetins

• Cytokinin ribosides
• Activation of A2A or A3 adenosine receptors
• Much more active agonists have already reached clinical trials

• Cytokinin ribosides
• Short plasmatic half-life (Mittelman et al. 1975)
• Problems with crossing biological barriers (Voller et al. 2010)
• Some cytokinin ribosides can be absorbed from the gut and even reach the brain
• Others are able to penetrate the skin
• Not guarantee similar behavior in humans because of differences in physiology
• Human skin has less hair follicles + thicker epidermis and dermis
• Once a cytokinin riboside is present in target skin layers
• Its pharmacokinetic properties may be seen as an advantage as they will prevent it from reaching the systemic circulation
• Limit possible side effects
• Vasodilatation in the case of A2AR agonists

• K, tZ and Pyratine
• Principle components of cosmetics products (Kinerase and Pyratine-6 lines)
• Beneficial effects on photoaging skin
• Ameliorated some symptoms of acne rosacea [1]

Inhibitors of cyclin-dependent kinases

• Olomoucine, bohemine, roscovitine were inspired by cytokinins (for a review see Jorda et al. 2012) [1]

KR and trans-zeatin ribosides (tZRs)

• Shown to have cytoprotective activities
• Can protect PC12 cells against serum starvation induced apoptosis (Lee et al. 2012)
• But not BAR
• Corresponding free bases
• TZ and K were inactive in this model
• Protective effects of KR were apparent at concentrations as low as 1 µM
• TZR did not show any significant activity at this concentration
• At a concentration of 100 µM - tZR was more effective than equimolar KR
• Therefore selected for follow-up experiments

Huntingtonova choroba

• TZRs protective activity in a serum deprivation model
• Mediated by activation of the A2A adenosine receptor (A2AR)
• Downstream protein kinase A (PKA)
• Co-treatment with inhibitors of those proteins abolished the protective effect
• A2AR has been suggested as a potential therapeutic target in Huntington’s disease
• Is highly expressed in the striatum, where mutant huntingtin causes early damage
• A2AR-selective agonists
• Effectively ameliorate several symptoms of Huntington’s disease in both cell cultures and animal models (Chou et al. 2005; Chiu et al. 2015)
• TZR’s ability to prevent huntingtin aggregation tested in PC12 cells overexpressing the mutant protein
• TZR treatment prevented both the mutant huntingtin aggregation and subsequent proteasome dysfunction in an A2AR and PKA dependent manner [1]

Para-topolin riboside (pTR, 6-(4-hydroxybenzylamino)purine riboside)

• A2AR agonist as well
• Isolated as a neuroprotective substance from the Chinese medicinal plant Gastrodia elata (Huang et al. 2011a; Huang et al. 2011b)
• PTR (designated as T1-11 in the study)
• Shown to interact with not only A2AR but also equilibrative nucleotide transporter ENT1
• ENT1 inhibition
• Can increase the amount of adenosine in extracellular space
• Possibly contribute to modulation of adenosinergic signaling in synapses where both proteins are present
• Treatment of R6/2 mice with pTR administered using a subcutaneous Alzet minipump for 48 h improved motor deterioration
• PTR administered in drinking water (0.05 mg/ml ad libitum, plasmatic or brain concentration unknown) improved the rotarod performance already after 2 weeks
• Effect persisted until the end of the experiment after 7 weeks
• Analysis of the brains showed a lower accumulation of huntingtin
• Improved activity of proteasome
• Higher levels of mRNA for brain-derived neurotrophic factor (BDNF)
• PTR also binds to A3R
• Another adenosine receptor with a known role in CNS physiology (Borea et al. 2014)
• Agonists of A3R have been shown to have neuroprotective effects in subarachnoid hemorrhage-induced brain damage (Maria Pugliese et al. 2007)
• Prolonged A3R activation may be neurotoxic (Luo et al. 2010)
• A3R agonistic activity of iPR has been reported (Blad et al. 2011) [1]

Immunomodulatory activities of cytokinins - tZR

• A2A is also expressed in various immune system cells
• T-lymphocytes, macrophages, monocytes and polymorphonuclear leukocytes
• Selective A2A receptor agonists
• Proposed as drugs for treatment of
• Graft-vs-host disease
• Colitis
• T-lymphocyte mediated ischemia-reperfusion injury (Chhabra et al. 2012; Jones and Kang 2015) [1]

• The immunomodulatory activity of tZR Lappas (2015)
• Inhibit production of interferon (IFN)-gamma, IL-2 and TNF-alpha
• In CD3+CD4+ or CD3+CD8+ T-lymphocytes incubated with anti-CD3 antibody
• In CD3+CD4+ population, tZR also inhibited production of IL-4
• EC50 values were in the range 10-100 µM
• TZR inhibited upregulation of CD25, CD69 and CD40L
• Inhibitory effects in the latter assays were strongly attenuated by co-treatment with 10 µM of the selective A2A receptor antagonist ZM241385
• Confirming its involvement
• In mice, tZR decreased the number of white-blood cells in intraperitoneal infiltrate in thioglycollate-induced peritonitis
• TZR was administered as a 1 mg/kg i.p. bolus at times 0, 1.5 and 3 h after the thioglycollate injection
• TZR is not a very potent immunomodulator in terms of the active concentration
• Absence of cytotoxic effects up to 1000 µM suggest that the low activity could be possibly compensated by the administration of higher doses.

KR

• May be a potential drug for the treatment of multiple myelomas
• Found to suppress cyclin D1 and D2 transcription
• Followed by arrest of the cell-cycle and selective apoptosis in tumor cells (Tiedemann et al. 2008)
• May be also effective against leukemia stem cells (McDermott et al. 2012)

Cytokinin bases K and tZ

• Delay the onset of several characteristics related to aging in human skin fibroblasts (Rattan and Clark 1994; Rattan and Sodagam 2005)
• TZ, K and its analogue Pyratine
• Principal components of cosmetics used for the treatment of aging skin [1]
• K and tZ also induce cellular antioxidant defense [1]

Kinetin - K

• First recognized as the substance responsible for the cytokinin activity of autoclaved herring sperm
• Occur naturally in
• Plant material (Ge et al. 2004)
• Human cells
• Human urine (Barciszewski et al. 1996; Barciszewski et al. 2000)
• Endogenous K is a result of oxidative DNA damage [1]

Zpomaluje stárnutí

• Able to increase the lifespan of invertebrates
• Skin fibroblasts cultured in the presence of kinetin or trans-zeatin
• Retain some characteristics of cells of lower passage [1]

• Several characteristics related to aging in human skin fibroblasts during serial passage in vitro (Rattan and Clark 1994)
• Size and morphology of fibroblasts passaged in the presence of K resembled those of the cells at lower passage numbers
• K decreased the number of actin stress fibers
• Autofluorescence due to accumulated lipofuscin was also less intense
• Optimal anti-aging effects observed at 80 µM concentration for both tZ and K
• Long-term cultivation experiments were enabled by the remarkably low toxicity of the cytokinins tested [1]

• Beneficial effects of several other cytokinin bases were reported in the next years:
• Active compounds include
• Para-topolin
• IP
• K derivative 6-furfurylamino-9-(tetrahydropyran-2-yl)purine (Pyratine-6, PRK-124) [1]

• K, tZ and Pyratine are the principal ingredients of several marketed cosmeceuticals [1]

• K effects related to aging are not limited to cells and tissues
• Dietary K has been shown to increase the life span of Zaprionus paravittiger fruit flies
• Prolonged the larval and pupal stages
• Reduced the age-specific death rates throughout the adult lifespan (Sharma et al. 1995)
• Effect was accompanied by enhanced catalase activity (Sharma et al. 1997)
• K and several other cytokinin derivatives
• Able to increase the lifespan of Caenorhabditis elegans (unpublished data) [1]

Anti-oxidant activity of cytokinins

• Barcizsewski et al. (1997) hypothesized that endogenous K may arise as a consequence of oxidative damage of DNA
• Proposed mechanism of K formation assumes that hydroxy radical attack at the 5' carbon of the deoxyribose residue yields furfural
• This aldehyde reacts with the amino group of adenine and
• After intramolecular rearrangement, the resulting Schiff base is reduced into K (Barciszewski et al. 1997) [1]

• Using 8-oxo-2' -deoxyguanosine (8-oxo-dG) as a marker for oxidative damage of DNA, Olsen et al. (1999) showed that
• K significantly protects DNA against reactive oxygen species (ROS) generated by the Fenton reaction in vitro
• Effect was dose dependent
• Maximum of about 50% protection observed at 100 µM K

Antiglykační protekce

• Shown to protect proteins against oxidative/glycoxidative damage
• More efficiently than adenine in several experimental systems in vitro (Olsen et al. 1999)
• Decreasing protein carbonylation in an iron/ascorbate system
• Prevented formation of advanced glycation end-product pentosidine and aggregation after incubation of proteins with sugars [1]
• Active K concentrations were in the range 50-200 µM (Verbeke et al. 2000)

Evaluace antioxidačních vlastností cytokininů K, BA, para-topolin and iP

• Evaluated by fluorimetric and spectrophotometric assays (Brizzolari et al. 2016)
• With the exception of BA, all the compounds showed significant activity in the oxygen radical absorbance capacity (ORAC) assay at 2.5 and 5 µM
• In the Trolox equivalence antioxidant capacity (TEAC) assay, only para-topolin (0.5 - 5 µM) was active
• Probably due to the presence of a phenolic hydroxyl
• 2-deoxyribose degradation assay
• All the compounds were able to react with hydroxyl radicals generated in the
• Higher activity of iP was ascribed to the presence of the double bond in the N6-side chain
• Electron spin resonance, Hsiao et al. (2003)
• Short pretreatment with K of 70 and 150 µM effectively inhibited hydroxyl radical formation in collagen-activated platelets
• Direct comparison of K with L-ascorbic acid, DL-alpha-tocopherol, DL-alpha lipoic acid, ubiquinone and idebenone (McDaniel et al. 2005)
• K quenched radicals generated through the photochemical excitation of water molecules effectively only at a concentration of 1 µM
• Effective concentrations of the other compounds, with the exception of DL-alpha lipoic acid, were one or two orders of magnitude lower
• K (100 µM) completely prevented oxidation of low density lipoproteins by Cu2+
• Other compounds decreased the production of lipid hydroxyperoxides in equimolar concentrations less efficiently
• Idebenone: 80%,
• DL-alpha-tocopherol: 50%,
• Ubiquinone: 26%,
• DL-alpha lipoic acid: 22%
• L-ascorbic acid: 15%
• Microsome oxidation (NADPH/ADP/Fe3+) assay
• Exceptional activity of K was not observed
• Considered to be a more realistic model of cell membrane peroxidation
• Activity of K was comparable with that of ubiquinone and L-ascorbic acid
• Reduction of malonyldialdehyde equivalents by 24-27%
• Other test compounds decreased MDA production by 47-55% [1]

Chelatace a SOD aktivita

• Cytokinins may also form complexes with ions of metals with the ability to quench ROS
• Superoxide dismutase-like activity of Cu2+ complexes of K and BA have been reported (Goldstein and Czapski 1991) [1]
• Cu2+ complexes of BA derivatives
• Protect against oxidative damage in vivo (aloxan induced diabetes) has been observed (Štarha et al. 2009) [1]

Activate cellular anti-oxidant defense mechanisms

• Induction of antioxidant enzymes may also contribute to the lifespan extension observed in flies after K treatment (Sharma et al. 1997) [1]
• Enhances the catalase activity in this organism
• Exhibit protective effects in the D-galactose model of glycoxidative stress
• Cultured rat astrocytes
• K partially reversed a decrease in the activities of glutathione peroxidase and superoxide dismutase induced by D-galactose treatment
• Decreased malonyldialdehyde concentration in the cell membranes
• Increased cell viability
• Active concentrations were in the range 50-100 µM
• Beneficial effects in rats receiving daily subcutaneous injections of D-galactose at 125 mg/kg for 6 weeks
• K (10, 20 and 40 mg/kg) was administered by gastric perfusion for the whole period of galactose exposition
• no information was provided about K concentrations in plasma or brain tissue
• A dose of 10 mg/kg was concluded to have the most promising effect
• Attenuate the negative effects of D-galactose in brain tissue on
• Malonyldialdehyde concentration
• Activities of glutathione peroxidase
• Superoxide dismutase (Liu et al. 2011) [1]

Cytoprotective activity of K

• Could be mediated by its metabolite KR and/or appropriate ribotides
• Conversion of cytokinin bases into their respective riboside 5’-monophosphates by human cells has been reported (Mlejnek and Doležel 2005)
• But tissues may differ in expression and/or activity of phosphoribosyltransferase
• Thus in the rate of K conversion into its riboside
• Hertz et al. (2013): conversion of K into its ribotides
• May be indeed important for its cytoprotective activity
• Mitoprotective PTEN-induced putative kinase 1 (PINK1, PARK6)
• Able to use kinetin riboside 5’-triphosphate (KRTP) as a donor of a phosphate group more efficiently than ATP
• Designated KRTP as a PINK1 neo-substrate
• K is converted into KRTP within the cells
• Treatment with K accelerated Parkin recruitment to depolarized mitochondria
• And suppressed oxidative stress-induced apoptosis in human-derived neural cells in a PINK1-dependent manner [1]

K a kůže

• K, and later tZ, was demonstrated for the first time in skin fibroblasts
• Cytokinin bases in skin protection in vitro and in vivo
• Protection against UV-induced damage
• Improved wound healing
• Aquaporin induction
• Modulate melanogenesis and keratinocyte differentiation
• Can improve multiple traits of photoaging skin
• Can improve some symptoms of acne rosacea
• McDaniel et al. (2005) compared the UV-protective activity of a group of antioxidants
• K
• L-ascorbic acid
• DL-alpha-tocopherol
• DL-alpha lipoic acid
• Ubiquinone
• Idebenone
• K was shown to protect primary keratinocytes against UVB (single dose, 200 mJ/cm2)
• K reduced the number of cells stained positively by immunohistochemistry with an antibody against thymine–dimer from 53% to 34%
• Idebenone, L-ascorbic acid and DL-alpha-tocopherol similar protection levels (29-35% of positive cells)
• Ubiquinone and DL lipoic acid were not active [1]

• UV-protective activity of the compounds was tested in patients
• Each compound on five subjects as ethanolic solutions (0.5% w/w) on mid-back regions once a day for 2 weeks
• Sunburn cells (SBC) induced by a 1.5x minimal erythema dose of UVB were quantified in biopsies obtained 20 h later
• K reduced the number of SBC by 20%
• 10% for ubiquinone and DL-lipoic
• Idebenone reduction by 38
• DL-alpha-tocopherol by 30%
• ascorbic acid did not have any protective effect
• no UV protective effects of K were observed in pigs (Tournas et al. 2006) [1]

• Cytokinin bases
• Promote differentiation of keratinocytes

Maturace - diferenciace keratinocytů

• K at 40 - 200 µM concentration induced growth arrest
• Changes of several markers of differentiation (keratin K10 and involucrin) in human keratinocytes in cell culture (Berge et al. 2006)
• Effect was augmented by the presence of Ca2+ ions
• Other markers of differentiation (trans-glutaminase) were unchanged
• K-induced differentiation might be mediated by pathways different from those activated by other differentiation inducing agents
• Treatment with K improved the sensitivity of aging keratinocytes to the differentiating effects of Ca2+ ions
• Effect was accompanied by induction of Hsp90, Hsp70 and heme-oxygenase-1
• V.s. mediated by stress-induced hormesis [1]

• To by se tedy mohlo dobře doplňovat s vitamínem D a kalciovou mastí při terapii třeba psoriázy ?

• Models using keratinocytes from psoriatic lesions
• Might be more appropriate for evaluation of the utility of cytokinins in the therapy of psoriasis [1]

• Positive effect of K on filaggrin levels
• Another marker of keratinocyte differentiation observed in an in vitro reconstructed skin equivalent (Vičanová et al. 2006)
• K promoted the growth of keratinocytes
• Increase in the number of Ki67-positive cells [1]

Therapy of pigmentation disorders

• pokud K snižovalo kumulaci lipofuscinu, bude to projasňovat stárnoucí kůži...

• K has been reported to decrease hyperpigmentation in dogs (Kimura and Doi 2004)

Topical K

• In multiple open-label single-arm clinical trials

Lotion 0.1% (Kinerase) 2xd

• Treatment for mildly to moderately photodamaged facial skin in 32 subjects
• 12 and 24 week treatments
• Improve significantly skin texture
• Mottled hyperpigmentation and fine wrinkles
• Improved transdermal water loss (McCullough and Weinstein 2002)
• Patients (N=17) with mild to moderate facial rosacea
• In a 12 week study (Wu et al. 2007)
• Reduced redness
• Statistically insignificant, effect on telangiectasia
• Almost 60 % of the subjects showed at least moderate improvement
• Generally well tolerated
• In rare cases, acne or a rash was observed after 8 weeks
• Wanitphakdeedecha et al. (2015)- on the photoaging facial skin of 100 Thai subjects
• 12 weeks’ treatment small but statistically significant improvements in overall skin condition
• Skin texture, color and wrinkles
• Improved ultraviolet spots and redness
• Chiu et al. (2007) evaluated possible synergistic effects of using a combination of K (0.03%) and niacin (4%) in a double-blind clinical study
• 52 Taiwanese subjects
• Serum containing either applied to one half of the face and the vehicle to the other twice daily for 12 weeks
• K and niacin (27 subjects)
• niacin alone (25 subjects)
• Combination of K and niacin had a larger positive effect on most of the evaluated parameters
• Corneal hydration
• Erythema index
• Differences between the treatments niacin + K x niacin alone = not statistically significant

Neuroprotective activity

• K protects rat astrocytes in vitro
• Protects rat brain against glycoxidative damage in the D-galactose model via promotion of anti-oxidant defense (Liu et al. 2011)
• KRTP increases kinase activity of PTEN-induced putative kinase 1 (PINK1)
• PINK1 is critical for mitochondrial quality control
• Accumulates in the outer membrane of impaired mitochondria
• Through the recruitment of Parkin protein
• Targets them for autophagy

• Both PINK1 and Parkin
• Mutated in the familial forms of recessive Parkinson’s disease

• Treatment with K
• Protected SH-S5Y5 neuroblastoma cells against proteasomal and oxidative stress in PINK1-dependent manner

• K is therefore an interesting drug candidate for the treatment of Parkinson’s disease
• Possibly also therapy of other diseases with mitochondrial dysfunction

• Other N6-substitued adenines might have similar type of activity
• May prove difficult:
• Base and riboside require multistep metabolic activation
• Have to mimic the shape and behavior of ATP in the PINK1 binding pocket (Hertz et al. 2013) [1]

• Candidate drug for neurodegenerative disease familial dysautonomia
• Clinical study (NCT02274051) is in the process of recruiting patients at the time of writing this text according to web page www.clinicaltrials.gov [1]

• In vitro and in vivo experiments
• K is able to correct aberrant splicing of pre-mRNA originating from the IKBKAP gene (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein)
• Very limited number of transcripts with splicing influenced by the treatment suggests that K interacts with regulators or components of specific spliceosome sub-species [1]

• Quench radicals directly
• Also been observed for several other cytokinin bases
• K modulates splicing of certain pre-RNAs (Slaugenhaupt et al. 2004)
• May also modulate splicing of transcripts of some genes related to cytoprotection.
• Cytoprotective activity of K, including its intracellular conversion into respective ribotides
• KRTP increases the activity of the mitoprotective kinase PINK1 by acting as a more active ATP analogue (neosubstrate)
• Ribosylation of cytokinin bases, cannot be ruled out

• Unique activities of K relevant for the treatment of
• Parkinson’s disease (PINK1 activation)
• Familial dysautonomia (correction of aberrant splicing of IKBKAP transcripts)
• Recruitment of patients for a study of familial dysautonomia is currently underway
• Therapy would require chronic administration of K [1]

N6-benzyladenine - BA

• Is a stimulator of melanogenesis (Kim et al. 2009) [1]
• 50 and 100 µM stimulated melanogenesis in B16 mouse melanoma cells via
• protein kinase A mediated induction of microphthalmia-associated transcription factor (MITF)
• Tyrosinase
• Tyrosinase-related proteins 1 and 2 (TYRP1, TYRP2) [1]
• BA activated protein kinase A in a cAMP independent manner [1]
• Para-topolin
• Induced tyrosinase expression and melanogenesis in B16 cells (unpublished data) [1]

• Takže ženy psychicky trpící z výskytu melasma na obličeji - nežádoucí pigmentací v obličeji - by se měli vhynout nadměrné konzumaci stravy s BA, para-topulinu a podobně účnikujícími chemikáliemi, stejně tak s inhibitory jaterní clarance melaninu, ženských hormonů a těchto rostlinných stimulátorů, popřípadě dalších.

Trans-zeatin

Skin

• Skin fibroblasts cultured in the presence of kinetin or trans-zeatin
• Retain some characteristics of cells of lower passage [1]
• TZ on in vitro také zpomaloval aging of a fibroblast population were reported more than 10 years later (Rattan and Sodagam 2005)
• TZ has been shown to induce hydrogen peroxide decomposing enzymes in human skin fibroblasts
• Treatment was also able to protect both proliferating and senescent cells against the hydrogen peroxide induced cell death (Rattan and Sodagam 2005) [1]
• TZ could improve skin hydration and wound healing
• Prevent detrimental effects of photoaging on those processes (Ji et al. 2010)[1]
• TZ at 40 and 80 µM concentration also induced expression of aquaporin 3 protein (AQP3) in spontaneously immortalized HaCaT keratinocytes
• Ameliorate a UV-induced decrease in AQP3 concentrations and membrane water permeability to a large extent
• Pharmacological MAPK pathway inhibitors revealed that tZ inhibits UV-induced MEK/ERK activation
• Promote wound healing in the scratch assay with either irradiated or non-irradiated cell cultures (Ji et al. 2010)
• Inhibit (tZ at 20-40 µM) UVB-induced MMP-1 expression in skin fibroblasts (Yang et al. 2009) [1]

Neuroprotection

• TZ also exhibits multiple activities relevant for the therapy of diseases of the central nervous system
• In particular Alzheimer’s disease
• Identified as the substance responsible for inhibition of rat acetylcholinesterase
• Contained in an extract from the traditional Korean edible plant Fiatoua villosa (IC50 1.09 × 10-4 M) (Heo et al. 2002) [1]
• TZ protects rat pheocytohroma cells PC12 against toxic effects of an amyloid beta fragment comprising amino acids 25–35
• TZ treatment had a positive effect on both ROS production and cell viability (Choi et al. 2009)
• Beneficial effect of long-term treatment with tZ on scopolamine-induced amnesia
• Scopolamine is an antagonist of muscarinic acetylcholine receptors
• Its administration also influences other neurotransmitter systems
• Repeated treatment of scopolamine has been shown to
• Decrease levels of monoamines
• noradrenaline, dopamine and serotonin
• Increase oxidative stress in brain (Haider et al. 2016) [1]
• Choi et al.’s study (2009), animals had ad libitum access to either normal drinking water
• Control
• Scopolamine control groups
• TZ solution (0.002%, 0.004%, and 0.008% w/v) for 21 days
• After teratment temporal amnesia was induced by a single s.c. injection of scopolamine (1 mg/kg)
• Behavioral tests were started 30 min later
• All 3 tested concentrations of fZ markedly attenuated negative effects of scopolamine on passive avoidance and spontaneous alternation behaviors in the Y-maze test
• Animals were sacrificed and acetylcholinesterase activity in brain lysates was measured
• Two highest tZ concentrations were able to reduce the effects of scopolamine on acetylcholinesterase activity
• Similar effects on behavior were observed when mice (Choi et al. 2009)

BARs

• High anticancer activity of BARs hydroxylated on the phenyl ring (Doležal et al. 2007; Voller et al. 2010)

iPR and BAR

• Activate the Nrf2 pathway
• Protect cells against oxidative stress (Dassano et al. 2014) [1]
• 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced superoxide production by HL-60 cells differentiated along the neutrophilic lineage
• Topical TPA-induced oxidative stress stimulates an inflammatory response
• Pretreatment with iPR and BAR
• Attenuated the inflammation
• Reduced the number of infiltrating neutrophils
• Nrf2 activation could be involved
• Authors discussed possible involvement of glucocorticoid receptor signaling
• IPR induced several transcripts in this pathway
• And adenosine receptor A3R activation
• A3R agonistic activity of iPR and tZR was reported recently (Blad et al. 2011)
• A3R regulates various aspects of inflammation
• Neutrophile chemotaxis
• Superoxide production (van der Hoeven et al. 2010) [1]
• Anti-inflammatory effects of A3R activation
• Via the inhibition of NF-kappa B dependent production of cytokines
• Including TNF-alpha
• A3R agonists have been shown to have robust anti-inflammatory activity in animal models of:
• Inflammatory bowel disease
• Systemic toxemia
• Pulmonary and liver inflammation
• Rheumatoid arthritis (Borea et al. 2014) [1]
• Nanomolar A3R agonist CF101 (IB-MECA)
• Structurally related to aromatic cytokinins
• Currently being evaluated as an antirheumatic and antipsoriatic agent in clinical trials
• Promising activity in patients with moderate to severe plaque psoriasis was reported (David et al. 2016) [1]

iPR

• Effects on natural killer cells (NK cells) (Ciaglia et al. 2014)
• At 10 µM concentration inhibited the cytotoxicity of NK cells against leukemia K562 cells
• Treatment prevented ERK/MAPK and STAT5 activation in IL-2-activated NK cells
• Downregulated the expression of activating receptors NKp44 and NKG2D
• Decreased secretion of cyto/chemokines
• RANTES, MIP-1?, TNF-alpha and IFN-gamma
• Topical application of iPR significantly reduced ear edema in a mouse model of croton oil-induced ear dermatitis
• With a potency comparable to that of indomethacin
• Histology analysis showed
• Lower leukocyte infiltration
• Decreased staining of the natural cytotoxicity receptor NKp46
• Expression is typical for NK cells in irritated mid and papillary dermis

• Purinergic activity
• Alone may not warrant future development of such compounds into drugs
• Other pathways could possibly be involved in immunomodulatory activity
• Activation of an Nrf2 response
• Inhibition of farnesyl diphosphate synthase
• Inhibition of protein farnesylation by iPR mediates:
• Anti-cancer and immunomodulatory activities (Ciaglia et al. 2013; Laezza et al. 2006)
• Ability to decrease the accumulation of abnormal lamin A (progerin) in the nuclear envelope of cells originating from patients with Hutchinson-Gilford progeria (Bifulco et al. 2013)
• Progerin resulting from aberrant processing of wild type lamin A transcripts accumulates in tissues
• Including skin of elderly
• Suggested as a biomarker of aging (McClintock et al. 2007)
• Topical application of iPR (or possibly iP) could have multiple benefits [1]

K derivative 6-furfurylamino-9-(tetrahydropyran-2-yl)purine (Pyratine-6, PRK-124)

• From Laboratory of Growth Regulators, Olomouc, Czech Republic
• Developed by companies Senetek PLC and Pyratine PLC, USA
• Aging and photodamaged skin of 40 subjects (34 finished the study)
• Pyratine (0.1%) was reported to improve skin
• Moisturization,
• Roughness,
• Mottled hyperpigmentation
• Fine wrinkles
• Facial erythema in comparison with the baseline within 4 weeks (McCullough et al. 2008)
• Application of the compound (0.125%) 2xd
• Also improved symptoms of acne rosacea x baseline in a 12 week study (Ortiz et al. 2009)
• 18 subjects were followed over an extended 48 week trial (Tremaine et al. 2010)
• 44 % reduction in erythema severity
• 89 % reduction in inflammatory lesions observed at week 48
• Positive effect on telangiectasias, transepidermal water loss and skin dryness
• No treatment-induced skin irritation was observed [1]

K and Pyratine

• Shown to have beneficial effects on acne rosacea
• Skin condition with an inflammatory component (Wu et al. 2007; Tremaine et al. 2010). [1]